ABSTRACT The mammalian mitochondrial ribosome is a ribonucleoprotein complex responsible for the synthesis of crucial polypeptides of the protein complexes required for oxidative phosphorylation, which generate about 90% of the energy currency (ATP) for the cell. An understanding of the structural and functional differences between bacterial and eukaryotic ribosomes is required for an effective treatment of bacterial infectious diseases. While bacterial ribosomes are the targets of several antibiotics, it is essential that the mitochondrial ribosomes (mitoribosomes) of the host cell not be susceptible to the toxic effects of the antibiotic. Furthermore, defects in mitochondrial translation are associated with several of the human mitochondrial diseases. The long-term goal of our study is to understand the mechanism of translation in mammalian mitochondria by determining structures for various functional complexes of the mitoribosome at atomic resolution. This knowledge will not only help us understand the genetic diseases related to mitochondrial translation, but will also facilitate the identification of drug targets specific to the bacterial system. Mammalian mitoribosomes are inherently poor candidates for crystallographic analysis, due to their compositional heterogeneity, and low abundances in the cell. On the other hand, an important recent technological advancement in the three-dimensional cryo-electron microscopy (3D cryo-EM) field has made it feasible to obtain close to atomic resolution structures of the macromolecular assemblies, thereby making it now the most powerful tool to study complex structures such as mitoribosomes. We will use state-of-the-art 3D cryo-EM to determine high-resolution (3.3 ? 5 ) structures of specific functional complexes of the mammalian mitoribosome that are formed during main translational events described under four Specific Aims, pertaining to translation (1) initiation, (2) elongation, (3) termination, and (4) mitoribosome recycling steps. These include complexes from our on-going studies, involving a mammalian mitochondrial translation initiation factor (IF2mt), an elongation factor (EF-G1mt), and two recycling factors (RRFmt and EF-G2mt). In addition, we will study structures of new complexes involving mitochondrial translational elongation factor EF-Tumt and the mitochondrial nascent-polypeptide release factor, RF1mt. Our strong progress and preliminary data suggest high feasibility of the proposed studies. The high-resolution cryo-EM maps generated for each of these complexes will allow us to directly compare specific steps of translation between the bacterial system (for which atomic structures of homologous complexes are known) and the mammalian mitochondrial system, and to identify potential drug targets.